Hyperactive piggybac transposases

ABSTRACT

The present invention provides PiggyBac transposase proteins, nucleic acids encoding the same, compositions comprising the same, kits comprising the same, non-human transgenic animals comprising the same, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/221,777, filedJul. 28, 2016, which is a continuation of U.S. Ser. No. 13/774,718,filed Feb. 22, 2013 (now, U.S. Pat. No. 9,546,382), which is adivisional application of U.S. Ser. No. 12/712,504, filed Feb. 25, 2010(now, U.S. Pat. No. 8,399,643), which claims priority to U.S.provisional application Ser. No. 61/155,804 filed Feb. 26, 2009, thecontents of each of which are incorporated herein by reference in theirentirety.

FIELD OF INVENTION

The present invention is directed, in part, to PiggyBac transposaseproteins, nucleic acids encoding the same, compositions comprising thesame, kits comprising the same, non-human transgenic animals, andmethods of using the same.

INCORPORATION OF THE SEQUENCE LISTING

The application contains, as a separate part of the disclosure, aSequence listing in computer-readable form named “POTH-013-C02US_SeqListing.txt” which was created on Dec. 20, 2017 and is 636 kilobytesin size, the contents of which are incorporated by reference herein intheir entirety.

BACKGROUND OF THE INVENTION

The PiggyBac (PB) transposase, part of a larger PB family oftransposases found in vertebrates and invertebrate PB transposons, is acompact functional transposase protein that catalyzes the excision andre-integration of the PB transposon (Fraser et al., Insect Mol. Biol.,1996, 5, 141-51; Mitra et al., EMBO J., 2008, 27, 1097-1109; and Ding etal., Cell, 2005, 122, 473-83). In many cases, an increase in themovement of the transposon to another part of the genome is desired. Forselection of a hyperactive transposase, one would want to select atransposase that can rapidly and efficiently mobilize a transposon fromone genomic location to another. Thus, for a particular experimentalperiod, a hyperactive transposase would be desired to yield a greaternumber of transposon integrations per cell versus a non-hyperactivetransposase. Provided herein are hyperactive PiggyBac transposaseproteins, nucleic acids encoding the same, compositions comprising thesame, kits comprising the same, non-human transgenic animals comprisingthe same, and methods of using the same.

SUMMARY OF THE INVENTION

The present invention provides proteins comprising at least 80% sequenceidentity to SEQ ID NO: 2, and comprising at least one of the followingamino acid substitutions in SEQ ID NO: 2: an asparagine for the serineat position 3; a valine for the isoleucine at 30 position 30; a serinefor the alanine at position 46; a threonine for the alanine at position46; a tryptophan for the isoleucine at position 82; a proline for theserine at position 103; a proline for the arginine at position 119; analanine for the cysteine at position 125; a leucine for the cysteine atposition 125; a serine for the glycine at position 165; a lysine for thetyrosine at position 177; a histidine for the tyrosine at position 177;a leucine for the phenylalanine at position 180; an isoleucine for thephenylalanine at position 180; a valine for the phenylalanine atposition 180; a leucine for the methionine at position 185; a glycinefor the alanine at position 187; a tryptophan for the phenylalanine atposition 200; a proline for the valine at position 207; a phenylalaninefor the valine at position 209; a phenylalanine for the methionine atposition 226; an arginine for the leucine at position 235; a lysine forthe valine at position 240; a leucine for the phenylalanine at position241; a lysine for the proline at position 243; a serine for theasparagine at position 258; a glutamine for the methionine at position282; a tryptophan for the leucine at position 296; a tyrosine for theleucine at position 296; a phenylalanine for the leucine at position296; a leucine for the methionine at position 298; an alanine for themethionine at position 298; a valine for the methionine at position 298;an isoleucine for the proline at position 311; a valine for the prolineat position 311; a lysine for the arginine at position 315; a glycinefor the threonine at position 319; an arginine for the tyrosine atposition 327; a valine for the tyrosine at position 328; a glycine forthe cysteine at position 340; a leucine for the cysteine at position340; a histidine for the aspartic acid at position 421; an isoleucinefor the valine at position 436; a tyrosine for the methionine atposition 456; a phenylalanine for the leucine at position 470; a lysinefor the serine at position 486; a leucine for the methionine at position503; an isoleucine for the methionine at position 503; a lysine for thevaline at position 552; a threonine for the alanine at position 570; aproline for the glutamine at position 591; or an arginine for theglutamine at position 591.

In some embodiments, the protein comprises at least 80% sequenceidentity to SEQ ID NO: 2, and comprises at least one of the followingamino acid substitutions in SEQ ID NO: 2: a serine for the glycine atposition 165; a leucine for the methionine at position 185; a glycinefor the alanine at position 187; a tryptophan for the phenylalanine atposition 200; a proline for the valine at position 207; a phenylalaninefor the methionine at position 226; a lysine for the valine at position240; a leucine for the phenylalanine at position 241; a glutamine forthe methionine at position 282; a tryptophan for the leucine at position296; a tyrosine for the leucine at position 296; a phenylalanine for theleucine at position 296; a leucine for the methionine at position 298;an alanine for the methionine at position 298; a valine for themethionine at position 298; an isoleucine for the proline at position311; a valine for the proline at position 311; a lysine for the arginineat position 315; an isoleucine for the valine at position 436; atyrosine for the methionine at position 456; a lysine for the serine atposition 486; a leucine for the methionine at position 503; or anisoleucine for the methionine at position 503.

In some embodiments, the protein comprises at least 80% sequenceidentity to SEQ ID NO: 2, and comprises at least one of the amino acidsubstitutions in SEQ ID NO: 2. In some embodiments, the proteincomprises at least 90% sequence identity to SEQ ID NO: 2, and comprisesat least one of the amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein comprises at least 95% sequence identity to SEQID NO: 2, and comprises at least one of the amino acid substitutions inSEQ ID NO: 2. In some embodiments, the protein comprises at least 99%sequence identity to SEQ ID NO: 2, and comprises at least one of theamino acid substitutions in SEQ ID NO: 2.

In some embodiments, the protein comprises at least 80% sequenceidentity to SEQ ID NO: 2, and comprises more than one of the amino acidsubstitutions in SEQ ID NO: 2. In some embodiments, the proteincomprises at least 90% sequence identity to SEQ ID NO: 2, and comprisesmore than one of the amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein comprises at least 95% sequence identity to SEQID NO: 2, and comprises more than one of the amino acid substitutions inSEQ ID NO: 2. In some embodiments, the protein comprises at least 99%sequence identity to SEQ ID NO: 2, and comprises more than one of theamino acid substitutions in SEQ ID NO: 2. In some embodiments, theprotein comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ IDNO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38,SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO:48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ IDNO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76,SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ IDNO:86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 96, SEQID NO: 98, SEQ ID NO: 100, SEQ ID NO:102, SEQ ID NO: 104, SEQ ID NO:106, or SEQ ID NO: 108.

In some embodiments, the protein comprises SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO:40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ IDNO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 62, SEQID NO: 76, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,or SEQ ID NO: 102.

The present invention also provides nucleic acids encoding any of theproteins described above. In some embodiments, the nucleic acidcomprises SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO:65, SEQ ID NO:67, SEQID NO: 69, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ IDNO:89, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, or SEQ ID NO: 107. In someembodiments, the nucleic acid comprises SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ IDNO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 61, SEQID NO: 75, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,or SEQ ID NO: 101.

The present invention also provides vectors comprising any of thenucleic acids described above encoding any of the proteins describedabove. In some embodiments, the vector is a plasmid. In someembodiments, the vector is a retrovirus. In some embodiments, theretrovirus comprises long terminal repeats, a psi packaging signal, acloning site, and a sequence encoding a selectable marker. The presentinvention also provides cells comprising any of the nucleic acids orvectors described herein. In some embodiments, the cell is a sperm or anegg.

The present invention also provides kits comprising: a vector comprisinga nucleic acid encoding any of the proteins described herein; and atransposon comprising an insertion site for an exogenous nucleic acid,wherein the insertion site is flanked by a first inverted repeatsequence comprising a sequence at least about 90% sequence identity toSEQ ID NO: 91 and/or a second inverted repeat sequence comprising asequence at least about 90% sequence identity to SEQ ID NO: 92.

The present invention also provides non-human, transgenic animalscomprising a nucleic acid molecule encoding any of the proteinsdescribed herein. In some embodiments, the non-human, transgenic animalfurther comprises a transposon comprising an insertion site for anexogenous nucleic acid, wherein the insertion site is flanked by a firstinverted repeat sequence comprising a sequence at least about 90%sequence identity to SEQ ID NO: 91 and/or a second inverted repeatsequence comprising a sequence at least about 90% sequence identity toSEQ ID NO: 92.

The present invention also provides methods of integrating an exogenousnucleic acid into the genome of at least one cell of a multicellular orunicellular organism comprising administering directly to themulticellular or unicellular organism: a transposon comprising theexogenous nucleic acid, wherein the exogenous nucleic acid is flanked bya first inverted repeat sequence comprising a sequence at least about90% sequence identity to SEQ ID NO: 91 and/or a second inverted repeatsequence comprising a sequence at least about 90% sequence identity toSEQ ID NO: 92; and a protein described herein to excise the exogenousnucleic acid from a plasmid, episome, or transgene and integrate theexogenous nucleic acid into the genome. In some embodiments, the proteinis administered as a nucleic acid encoding the protein. In someembodiments, the transposon and nucleic acid encoding the protein arepresent on separate vectors. In some embodiments, the transposon andnucleic acid encoding the protein are present on the same vector. Insome embodiments, the multicellular or unicellular organism is avertebrate. In some embodiments, the vertebrate animal is a mammal. Insome embodiments, the administering is administering systemically. Insome embodiments, the exogenous nucleic acid comprises a gene.

The present invention also provides methods of generating a non-human,transgenic animal comprising a germline mutation comprising: breeding afirst non-human, transgenic animal comprising a transposon with a secondnon-human, transgenic animal comprising a vector comprising a nucleotidesequence encoding any of the proteins described herein.

The present invention also provides methods of generating a non-human,transgenic animal comprising: introducing a nucleic acid moleculeencoding any of the proteins described herein into a cell underconditions sufficient to generate a transgenic animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pair of schematic diagrams depicting BII-sd2GFP andBII-sMdr1 PolyA trap transposons.

FIG. 2A-B is a schematic diagram and a graph depicting a transpositionanalysis by FACS.

FIG. 3A-C is a series of schematic diagrams depicting hyperactivetransposases.

FIG. 4A-C is a series of schematic diagrams depicting hyperactivetransposases.

FIG. 5 is a pair of graphs depicting a flow-cytometry assay of somehyperactive transposases.

FIG. 6A-B is a pair of graphs depicting the identification ofhyperactive transposases by flow-cytometry analysis.

DESCRIPTION OF EMBODIMENTS

The present invention provides hyperactive PiggyBac transposaseproteins. In some embodiments, the protein comprises at least 75%sequence identity to SEQ ID NO: 2, and comprises a substitution in atleast one of the following amino acid positions in SEQ ID NO: 2:position 3, position 30, position 46, position R2, position 103,position 119, position 125, position 165, position 177, position 180,position 185, position 187, position 200, position 207, position 209,position 226, position 235, position 240, position 241, position 243,position 258, position 282, position 296, position 298, position 311,position 315, position 319, position 327, position 328, position 340,position 421, position 436, position 456, position 470, position 486,position 503, position 552; position 570, or position 591.

In some embodiments, the protein comprises at least 75% sequenceidentity to SEQ ID NO: 2, and comprises a conservative amino acidsubstitution in at least one of the following amino acid positions inSEQ ID NO: 2: position 3, position 30, position 46, position 82,position 103, position 119, position 125, position 165, position 177,position 180, position 185, position 187, position 200, position 207,position 209, position 226, position 235, position 240, position 241,position 243, position 258, position 282, position 296, position 298,position 311, position 315, position 319, position 327, position 328,position 340, position 421, position 436, position 456, position 470,position 486, position 503, position 552; position 570, or position 591.

In some embodiments, the protein comprises at least 75% sequenceidentity to SEQ ID NO: 2, and comprises at least one of the followingamino acid substitutions in SEQ ID NO: 2: an asparagine for the serineat position 3; a valine for the isoleucine at position 30; a serine forthe alanine at position 46; a threonine for the alanine at position 46;a tryptophan for the isoleucine at position 82; a proline for the serineat position 103; a proline for the arginine at position 119; an alaninefor the cysteine at position 125; a leucine for the cysteine at position125; a serine for the glycine at position 165; a lysine for the tyrosineat position 177; a histidine for the tyrosine at position 177; a leucinefor the phenylalanine at position 180; an isoleucine for thephenylalanine at position 180; a valine for the phenylalanine atposition 180; a leucine for the methionine at position 185; a glycinefor the alanine at position 187; a tryptophan for the phenylalanine atposition 200; a proline for the valine at position 207; a phenylalaninefor the valine at position 209; a phenylalanine for the methionine atposition 226; an arginine for the leucine at position 235; a lysine forthe valine at position 240; a leucine for the phenylalanine at position241; a lysine for the proline at position 243; a serine for theasparagine at position 258; a glutamine for the methionine at position282; a tryptophan for the leucine at position 296; a tyrosine for theleucine at position 296; a phenylalanine for the leucine at position296; a leucine for the methionine at position 298; an alanine for themethionine at position 298; a valine for the methionine at position 298;an isoleucine for the proline at position 311; a valine for the prolineat position 311; a lysine for the arginine at position 315; a glycinefor the threonine at position 319; an arginine for the tyrosine atposition 327; a valine for the tyrosine at position 328; a glycine forthe cysteine at position 340; a leucine for the cysteine at position340; a histidine for the aspartic acid at position 421; an isoleucinefor the valine at position 436; a tyrosine for the methionine atposition 456; a phenylalanine for the leucine at position 470; a lysinefor the serine at position 486; a leucine for the methionine at position503; an isoleucine for the methionine at position 503; a lysine for thevaline at position 552; a threonine for the alanine at position 570; aproline for the glutamine at position 591; or an arginine for theglutamine at position 591.

In some embodiments, the protein comprises at least 75% sequenceidentity to SEQ ID NO: 2, and comprises a substitution in at least oneof the following amino acid positions in SEQ ID NO: 2: position 165,position 185, position 187, position 200, position 207, position 226,position 240, position 241, position 282, position 296, position 298,position 311, position 315, position 436, position 456, position 486, orposition 503.

In some embodiments, the protein comprises at least 75% sequenceidentity to SEQ ID NO: 2, and comprises a conservative amino acidsubstitution in at least one of the following amino acid positions inSEQ ID NO: 2: position 165, position 185, position 187, position 200,position 207, position 226, position 240, position 241, position 2 2,position 296, position 298, position 311, position 315, position 436,position 456, position 486, or position 503.

In some embodiments, the protein comprises at least 75% sequenceidentity to SEQ ID NO: 2, and comprises at least one of the followingamino acid substitutions in SEQ ID NO: 2: a serine for the glycine atposition 165; a leucine for the methionine at position 185; a glycinefor the alanine at position 187; a tryptophan for the phenylalanine atposition 200; a proline for the valine at position 207; a phenylalaninefor the methionine at position 226; a lysine for the valine at position240; a leucine for the phenylalanine at position 241; a glutamine forthe methionine at position 282; a tryptophan for the leucine at position296; a tyrosine for the leucine at position 296; a phenylalanine for theleucine at position 296; a leucine for the methionine at position 298;an alanine for the methionine at position 298; a valine for themethionine at position 298; an isoleucine for the proline at position311; a valine for the proline at position 311; a lysine for the arginineat position 315; an isoleucine for the valine at position 436; atyrosine for the methionine at position 456; a lysine for the serine atposition 486; a leucine for the methionine at position 503; or anisoleucine for the methionine at position 503.

In some embodiments, the protein (as nucleic acid, as nucleic acid in avector, or as purified recombinant protein) comprises at least 80%sequence identity to SEQ ID NO: 2, and comprises at least one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 85% sequenceidentity to SEQ ID NO: 2, and comprises at least one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 90% sequenceidentity to SEQ ID NO: 2, and comprises at least one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 95% sequenceidentity to SEQ ID NO: 2, and comprises at least one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 99% sequenceidentity to SEQ ID NO: 2, and comprises at least one of theaforementioned amino acid substitutions in SEQ ID NO: 2.

In some embodiments, the protein (as nucleic acid, as nucleic acid in avector, or as purified recombinant protein) comprises at least 75%sequence identity to SEQ ID NO: 2, and comprises more than one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 80% sequenceidentity to SEQ ID NO: 2, and comprises more than one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 85% sequenceidentity to SEQ ID NO: 2, and comprises more than one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 90% sequenceidentity to SEQ ID NO: 2, and comprises more than one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 95% sequenceidentity to SEQ ID NO: 2, and comprises more than one of theaforementioned amino acid substitutions in SEQ ID NO: 2. In someembodiments, the protein (as nucleic acid, as nucleic acid in a vector,or as purified recombinant protein) comprises at least 99% sequenceidentity to SEQ ID NO: 2, and comprises more than one of theaforementioned amino acid substitutions in SEQ ID NO: 2.

As used herein, “sequence identity” is determined by using thestand-alone executable BLAST engine program for blasting two sequences(b12seq), which can be retrieved from the National Center forBiotechnology Information (NCBI) ftp site, using the default parameters(Tatusova and Madden, FEMS Microbial Lett., 1999, 174, 247-250; which isincorporated herein by reference in its entirety).

As used herein, “conservative” amino acid substitutions may be definedas set out in Tables A, B, or C below. Hyperactive transposases includethose wherein conservative substitutions have been introduced bymodification of polynucleotides encoding polypeptides of the invention.Amino acids can be classified according to physical properties andcontribution to secondary and tertiary protein structure. A conservativesubstitution is recognized in the art as a substitution of one aminoacid for another amino acid that has similar properties. Exemplaryconservative substitutions are set out in Table A.

TABLE A Conservative Substitutions I Side Chain Characteristics AminoAcid Aliphatic Non-polar G A P I L V F Polar—uncharged C S T M N QPolar—charged D E K R Aromatic H F W Y Other N Q D E

Alternately, conservative amino acids can be grouped as described inLehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY,N.Y. (1975), pp. 71-77) as set forth in Table B.

TABLE B Conservative Substitutions II Side Chain Characteristic AminoAcid Non-polar (hydrophobic) Aliphatic: A L I V P Aromatic: F W YSulfur-containing M Borderline G Y Uncharged-polar Hydroxyl: S T YAmides: N Q Sulfhydryl: C Borderline: G Y Positively-charged (Basic) K RH Negatively-charged (Acidic) D E

Alternately, exemplary conservative substitutions are set out in TableC.

TABLE C Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val Leu Ile Met Arg (R) Lys His Asn (N) Gln Asp (D)Glu Cys (C) Ser Thr Gln (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His(H) Lys Arg Ile (I) Leu Val Met Ala Phe Leu (L) Ile Val Met Ala Phe Lys(K) Arg His Met (M) Leu Ile Val Ala Phe (F) Trp Tyr Ile Pro (P) Gly AlaVal Leu Ile Ser (S) Thr Thr (T) Ser Trp (W) Tyr Phe Ile Tyr (Y) Trp PheThr Ser Val (V) Ile Leu Met Ala

It should be understood that the hyperactive PiggyBac transposasesdescribed herein are intended to include polypeptides bearing one ormore insertions, deletions, or substitutions, or any combinationthereof, of amino acid residues as well as modifications other thaninsertions, deletions, or substitutions of amino acid residues.

As used herein, “more than one” of the aforementioned amino acidsubstitutions means 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the recited aminoacid substitutions. In some embodiments, “more than one” means 2, 3, 4,or 5 of the recited amino acid substitutions. In some embodiments, “morethan one” means 2, 3, or 4 of the recited amino acid substitutions. Insome embodiments, “more than one” means 2 or 3 of the recited amino acidsubstitutions. In some embodiments, “more than one” means 2 of therecited amino acid substitutions.

In some embodiments, the protein comprises SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO: 2X, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:94, SEQ ID NO:96, SEQ IDNO:98, SEQ ID NO: 100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, orSEQ ID NO:108.

In some embodiments, the protein comprises SEQ ID NO: 24, SEQ ID NO: 26,SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:40,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:76,SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, or SEQ ID NO:102

The present invention also provides nucleic acids encoding any one ofthe hyperactive PiggyBac transposase proteins described herein. Thus,the present invention provides nucleic acids encoding a protein thatcomprises at least 75% (or 80%, 85%, 90%, 95%, or 99%) sequence identityto SEQ ID NO: 2, and comprises at least one of the following amino acidsubstitutions in SEQ ID NO: 2: an asparagine for the serine at position3; a valine for the isoleucine at position 30; a serine for the alanineat position 46; a threonine for the alanine at position 46; a tryptophanfor the isoleucine at position 82; a proline for the serine at position103; a proline for the arginine at position 119; an alanine for thecysteine at position 125; a leucine for the cysteine at position 125; aserine for the glycine at position 165; a lysine for the tyrosine atposition 177; a histidine for the tyrosine at position 177; a leucinefor the phenylalanine at position 180; an isoleucine for thephenylalanine at position 180; a valine for the phenylalanine atposition 180; a leucine for the methionine at position 185; a glycinefor the alanine at position 187; a tryptophan for the phenylalanine atposition 200; a proline for the valine at position 207; a phenylalaninefor the valine at position 209; a phenylalanine for the methionine atposition 226; an arginine for the leucine at position 235; a lysine forthe valine at position 240; a leucine for the phenylalanine at position241; a lysine for the proline at position 243; a serine for theasparagine at position 258; a glutamine for the methionine at position282; a tryptophan for the leucine at position 296; a tyrosine for theleucine at position 296; a phenylalanine for the leucine at position296; a leucine for the methionine at position 298; an alanine for themethionine at position 298; a valine for the methionine at position 298;an isoleucine for the proline at position 311; a valine for the prolineat position 311; a lysine for the arginine at position 315; a glycinefor the threonine at position 319; an arginine for the tyrosine atposition 327; a valine for the tyrosine at position 328; a glycine forthe cysteine at position 340; a leucine for the cysteine at position340; a histidine for the aspartic acid at position 421; an isoleucinefor the valine at position 436; a tyrosine for the methionine atposition 456; a phenylalanine for the leucine at position 470; a lysinefor the serine at position 486; a leucine for the methionine at position503; an isoleucine for the methionine at position 503; a lysine for thevaline at position 552; a threonine for the alanine at position 570; aproline for the glutamine at position 591; or an arginine for theglutamine at position 591.

In some embodiments, the nucleic acid encodes a protein that comprisesat least 75% (or 80%, 85%, 90%, 95%, or 99%) sequence identity to SEQ IDNO: 2, and comprises at least one of the following amino acidsubstitutions in SEQ ID NO: 2: a serine for the glycine at position 165;a leucine for the methionine at position 185; a glycine for the alanineat position 187; a tryptophan for the phenylalanine at position 200; aproline for the valine at position 207; a phenylalanine for themethionine at position 226; a lysine for the valine at position 240; aleucine for the phenylalanine at position 241; a glutamine for themethionine at position 282; a tryptophan for the leucine at position296; a tyrosine for the leucine at position 296; a phenylalanine for theleucine at position 296; a leucine for the methionine at position 298;an alanine for the methionine at position 298; a valine for themethionine at position 298; an isoleucine for the proline at position311; a valine for the proline at position 311; a lysine for the arginineat position 315; an isoleucine for the valine at position 436; atyrosine for the methionine at position 456; a lysine for the serine atposition 486; a leucine for the methionine at position 503; or anisoleucine for the methionine at position 503.

Given the redundancy in the genetic code, one skilled in the art couldgenerate numerous nucleotide sequences that encode any particularprotein. All such nucleotides sequences are contemplated herein. In someembodiments, the nucleic acid comprises SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:107. Insome embodiments, the nucleic acid comprises SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:33, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:61, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, or SEQ IDNO: 101.

The present invention also provides vectors comprising any of theaforementioned nucleic acids. Thus, the present invention providesvectors comprising a nucleic acid that encodes a protein that comprisesat least 75% (or 80%, 85%, 90%, 95%, or 99%) sequence identity to SEQ IDNO: 2, and comprises at least one of the following amino acidsubstitutions in SEQ ID NO: 2: an asparagine for the serine at position3; a valine for the isoleucine at position 30; a serine for the alanineat position 46; a threonine for the alanine at position 46; a tryptophanfor the isoleucine at position 82; a proline for the serine at position103; a proline for the arginine at position 119; an alanine for thecysteine at position 125; a leucine for the cysteine at position 125; aserine for the glycine at position 165; a lysine for the tyrosine atposition 177; a histidine for the tyrosine at position 177; a leucinefor the phenylalanine at position 180; an isoleucine for thephenylalanine at position 180; a valine for the phenylalanine atposition 180; a leucine for the methionine at position 185; a glycinefor the alanine at position 187; a tryptophan for the phenylalanine atposition 200; a proline for the valine at position 207; a phenylalaninefor the valine at position 209; a phenylalanine for the methionine atposition 226; an arginine for the leucine at position 235; a lysine forthe valine at position 240; a leucine for the phenylalanine at position241; a lysine for the proline at position 243; a serine for theasparagine at position 258; a glutamine for the methionine at position282; a tryptophan for the leucine at position 296; a tyrosine for theleucine at position 296; a phenylalanine for the leucine at position296; a leucine for the methionine at position 298; an alanine for themethionine at position 298; a valine for the methionine at position 298;an isoleucine for the proline at position 311; a valine for the prolineat position 311; a lysine for the arginine at position 315; a glycinefor the threonine at position 319; an arginine for the tyrosine atposition 327; a valine for the tyrosine at position 328; a glycine forthe cysteine at position 340; a leucine for the cysteine at position340; a histidine for the aspartic acid at position 421; an isoleucinefor the valine at position 436; a tyrosine for the methionine atposition 456; a phenylalanine for the leucine at position 470; a lysinefor the serine at position 486; a leucine for the methionine at position503; an isoleucine for the methionine at position 503; a lysine for thevaline at position 552; a threonine for the alanine at position 570; aproline for the glutamine at position 591; or an arginine for theglutamine at position 591.

In some embodiments, the vector comprises a nucleic acid that encodes aprotein that comprises at least 75% (or 80%, 85%, 90%, 95%, or 99%)sequence identity to SEQ ID NO: 2, and comprises at least one of thefollowing amino acid substitutions in SEQ ID NO: 2: a serine for theglycine at position 165; a leucine for the methionine at position 185; aglycine for the alanine at position 187; a tryptophan for thephenylalanine at position 200; a proline for the valine at position 207;a phenylalanine for the methionine at position 226; a lysine for thevaline at position 240; a leucine for the phenylalanine at position 241;a glutamine for the methionine at position 282; a tryptophan for theleucine at position 296; a tyrosine for the leucine at position 296; aphenylalanine for the leucine at position 296; a leucine for themethionine at position 298; an alanine for the methionine at position298; a valine for the methionine at position 298; an isoleucine for theproline at position 311; a valine for the proline at position 311; alysine for the arginine at position 315; an isoleucine for the valine atposition 436; a tyrosine for the methionine at position 456; a lysinefor the serine at position 486; a leucine for the methionine at position503; or an isoleucine for the methionine at position 503.

In some embodiments, the vector comprises a nucleic acid that comprisesSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ IDNO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ IDNO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:93, SEQ IDNO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQID NO: 105, or SEQ ID NO: 107. In some embodiments, the vector comprisesa nucleic acid that comprises SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:61, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:101.

In some embodiments, the vector is a plasmid. In other embodiments, thevector is a retrovirus. In some embodiments, the vector is a linear DNAmolecule. In some embodiments, the retrovirus comprises long terminalrepeats, a psi packaging signal, a cloning site, and a sequence encodinga selectable marker. In some embodiments, the vector is a viral vector,such as pLXIN (Clontech). The present invention also provides cells ororganisms comprising any of the aforementioned nucleic acids. Thus, thepresent invention provides cells or organisms comprising a nucleic acidthat encodes a protein that comprises at least 75% (or 80%, 85%, 90%,95%, or 99%) sequence identity to SEQ ID NO: 2, and comprises at leastone of the following amino acid substitutions in SEQ ID NO: 2: anasparagine for the serine at position 3; a valine for the isoleucine atposition 30; a serine for the alanine at position 46; a threonine forthe alanine at position 46; a tryptophan for the isoleucine at position82; a proline for the serine at position 103; a proline for the arginineat position 119; an alanine for the cysteine at position 125; a leucinefor the cysteine at position 125; a serine for the glycine at position165; a lysine for the tyrosine at position 177; a histidine for thetyrosine at position 177; a leucine for the phenylalanine at position180; an isoleucine for the phenylalanine at position 180; a valine forthe phenylalanine at position 180; a leucine for the methionine atposition 185; a glycine for the alanine at position 187; a tryptophanfor the phenylalanine at position 200; a proline for the valine atposition 207; a phenylalanine for the valine at position 209; aphenylalanine for the methionine at position 226; an arginine for theleucine at position 235; a lysine for the valine at position 240; aleucine for the phenylalanine at position 241; a lysine for the prolineat position 243; a serine for the asparagine at position 258; aglutamine for the methionine at position 282; a tryptophan for theleucine at position 296; a tyrosine for the leucine at position 296; aphenylalanine for the leucine at position 296; a leucine for themethionine at position 298; an alanine for the methionine at position298; a valine for the methionine at position 298; an isoleucine for theproline at position 311; a valine for the proline at position 311; alysine for the arginine at position 315; a glycine for the threonine atposition 319; an arginine for the tyrosine at position 327; a valine forthe tyrosine at position 328; a glycine for the cysteine at position340; a leucine for the cysteine at position 340; a histidine for theaspartic acid at position 421; an isoleucine for the valine at position436; a tyrosine for the methionine at position 456; a phenylalanine forthe leucine at position 470; a lysine for the serine at position 486; aleucine for the methionine at position 503; an isoleucine for themethionine at position 503; a lysine for the valine at position 552; athreonine for the alanine at position 570; a proline for the glutamineat position 591; or an arginine for the glutamine at position 591.

In some embodiments, the cells or organisms comprise a nucleic acid thatencodes a protein that comprises at least 75% (or 80%, 85%, 90%, 95%, or99%) sequence identity to SEQ ID NO: 2, and comprises at least one ofthe following amino acid substitutions in SEQ ID NO: 2: a serine for theglycine at position 165; a leucine for the methionine at position 185; aglycine for the alanine at position 187; a tryptophan for thephenylalanine at position 200; a proline for the valine at position 207;a phenylalanine for the methionine at position 226; a lysine for thevaline at position 240; a leucine for the phenylalanine at position 241;a glutamine for the methionine at position 282; a tryptophan for theleucine at position 296; a tyrosine for the leucine at position 296; aphenylalanine for the leucine at position 296; a leucine for themethionine at position 298; an alanine for the methionine at position298; a valine for the methionine at position 298; an isoleucine for theproline at position 311; a valine for the proline at position 311; alysine for the arginine at position 315; an isoleucine for the valine atposition 436; a tyrosine for the methionine at position 456; a lysinefor the serine at position 486; a leucine for the methionine at position503; or an isoleucine for the methionine at position 503.

In some embodiments, the cells or organisms comprise a nucleic acid thatcomprises SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ IDNO:103, SEQ ID NO:105, or SEQ ID NO:107. In some embodiments, the cellsor organisms comprise a nucleic acid that comprises SEQ ID NO: 23, SEQID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:33, SEQ ID NO:37, SEQID NO:39, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:61, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, or SEQ IDNO:101.

In some embodiments, the cell comprises any of the aforementionedvectors.

The present invention also provides kits comprising: 1) any of theaforementioned vectors; and 2) any of the hyperactive PiggyBactransposons described herein comprising an insertion site for anexogenous nucleic acid, wherein the insertion site is flanked by eitherone or more of the inverted repeat sequences that are specificallyrecognized by any of the aforementioned proteins. In some embodiments,the inverted repeats comprises a first inverted repeat and/or a secondinverted repeat, wherein the first inverted repeat comprises a sequenceat least about 80% sequence identity to SEQ ID NO: 91 and the secondinverted repeat sequence comprises a sequence at least about 80%sequence identity to SEQ ID NO: 92. In some embodiments, the firstinverted repeat comprises a sequence at least about 85% sequenceidentity to SEQ ID NO: 91 and the second inverted repeat sequencecomprises a sequence at least about 85% sequence identity to SEQ ID NO:92. In some embodiments, the first inverted repeat comprises a sequenceat least about 90% sequence identity to SEQ ID NO: 91 and the secondinverted repeat sequence comprises a sequence at least about 90%sequence identity to SEQ ID NO: 92. In some embodiments, the firstinverted repeat comprises a sequence at least about 95% sequenceidentity to SEQ ID NO: 91 and the second inverted repeat sequencecomprises a sequence at least about 95% sequence identity to SEQ ID NO:92. In some embodiments, the first inverted repeat comprises a sequenceat least about 99% sequence identity to SEQ ID NO: 91 and the secondinverted repeat sequence comprises a sequence at least about 99%sequence identity to SEQ ID NO: 92. In some embodiments, the firstinverted repeat comprises a sequence identical to SEQ ID NO: 91 and thesecond inverted repeat sequence comprises a sequence identical to SEQ IDNO: 92.

As stated above, the aforementioned transposon is a nucleic acid that isflanked at either end by inverted repeats which are recognized by anenzyme having PiggyBac transposase activity. By “recognized” is meantthat a PiggyBac transposase, such as any of the aforementioned proteins,is capable of binding to the inverted repeat, excising the segment ofnucleic acid flanked by the inverted repeats, and integrating thesegment of nucleic acid flanked by the inverted repeats into the genomeof the target cell.

In some embodiments, the left (5′) inverted repeat sequence is:5′-CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG-3′ (SEQ ID NO: 91) and the right(3′) inverted repeat is:5′-CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGC GTAAAATTGACGCATG-3′(SEQ ID NO: 92).

The various elements of the transposon systems described herein can beproduced by standard methods of restriction enzyme cleavage, ligation,and molecular cloning. One protocol for constructing the vectorsdescribed herein includes the following steps. Purified nucleic acidfragments containing the desired component nucleotide sequences as wellas extraneous sequences are cleaved with restriction endonucleases frominitial sources, such as a vector comprising the PiggyBac transposasegene. Fragments containing the desired nucleotide sequences areseparated from unwanted fragments of different size using conventionalseparation methods, such as for example, agarose gel electrophoresis.The desired fragments are excised from the gel and ligated together inthe appropriate configuration so that a circular nucleic acid or plasmidcontaining the desired sequences, such as for example, sequencescorresponding to the various elements of the subject vectors, asdescribed above is produced. Where desired, the circular molecules soconstructed are then amplified in a prokaryotic host, such as forexample, E. coli. Alternately, an RNA comprising the PiggyBactransposase can be produced with an RNA polymerase, using a DNA plasmidas a substrate. Recombinant protein comprising the PiggyBac transposasecan be produced by methods including, but not limited to, in vitrotranscription and translation, or expression in E. coli followed bypurification by affinity or fractionation. The procedures of cleavage,plasmid construction, cell transformation, plasmid production, RNAtranscription/purification, and recombinant protein purificationinvolved in these steps are well known to one skilled in the art and theenzymes required for restriction and ligation are availablecommercially. Preparation of a PiggyBac transposon system is disclosedin, for example, WO 20061122442. Synthesis of at least one of thesequences described herein was generated by GeneArt AG (Regensberg,Germany).

The PiggyBac transposons described herein can include a wide variety ofinserted nucleic acids, where the nucleic acids can include a sequenceof bases that is endogenous and/or exogenous to a multicellular orunicellular organism. The nature of the nucleic acid can vary dependingupon the particular protocol being carried out. In some embodiments, theexogenous nucleic acid can be a gene. The inserted nucleic acid that ispositioned between the flanking inverted repeats can vary greatly insize. The only limitation on the size of the inserted nucleic acid isthat the size should not be so great as to inactivate the ability of thetransposon system to integrate the transposon into the target genome.The upper and lower limits of the size of inserted nucleic acid can bedetermined empirically by those of skill in the art.

In some embodiments, the inserted nucleic acid comprises at least onetranscriptionally active gene, which is a coding sequence that iscapable of being expressed under intracellular conditions, e.g. a codingsequence in combination with any requisite expression regulatoryelements that are required for expression in the intracellularenvironment of the target cell whose genome is modified by integrationof the transposon. The transcriptionally active genes of the transposoncan comprise a domain of nucleotides, i.e., an expression module thatincludes a coding sequence of nucleotides operably linked with requisitetranscriptional mediation or regulatory element(s). Requisitetranscriptional mediation elements that may be present in the expressionmodule include, but are not limited to, promoters, enhancers,termination and polyadenylation signal elements, splicing signalelements, and the like.

In some embodiments, the expression module includes transcriptionregulatory elements that provide for expression of the gene in a broadhost range. A variety of such combinations are known, where specifictranscription regulatory elements include, but are not limited to: SV40elements, transcription regulatory elements derived from the LTR of theRous sarcoma virus, transcription regulatory elements derived from theLTR of human cytomegalovirus (CMV), hsp70 promoters, and the like.

In some embodiments, at least one transcriptionally active gene orexpression module present in the inserted nucleic acid acts as aselectable marker. A variety of different genes have been employed asselectable markers, and the particular gene employed in the vectorsdescribed herein as a selectable marker is chosen primarily as a matterof convenience. Known selectable marker genes include, but are notlimited to: thymidine kinase gene, dihydrofolate reductase gene,xanthine-guanine phosphoribosyl transferase gene, CAD, adenosinedeaminase gene, asparagine synthetase gene, numerous antibioticresistance genes (tetracycline, ampicillin, kanamycin, neomycin, and thelike), aminoglycoside phosphotransferase genes, hygromycin Bphosphotransferase gene, and genes whose expression provides for thepresence of a detectable product, either directly or indirectly, suchas, for example, beta-galactosidase, GFP, and the like.

In addition to the at least one transcriptionally active gene, theportion of the transposon containing the inverted repeats also comprisesat least one restriction endonuclease recognized site, e.g. restrictionsite, located between the flanking inverted repeats, which serves as asite for insertion of an exogenous nucleic acid. A variety ofrestriction sites are known in the art and include, but are not limitedto: HindIII, PstI, SalI, AccI, HincII, XbaI, BamHI, SmaI, XmaI, KpnI,SacI, EcoRI, and the like. In some embodiments, the vector includes apolylinker, i.e. a closely arranged series or array of sites recognizedby a plurality of different restriction enzymes, such as those listedabove. In other embodiments, the inserted exogenous nucleic acid couldcomprise recombinase recognition sites, such as LoxP, FRT, or AttB/AttPsites, which are recognized by the Cre, Flp, and PhiC31 recombinases,respectively.

Where the source of hyperactive transposase is a nucleic acid thatencodes the hyperactive transposase, the nucleic acid encoding thehyperactive transposase protein is generally part of an expressionmodule, as described above, where the additional elements provide forexpression of the transposase as required.

The present invention also provides methods of integrating an exogenousnucleic acid into the genome of at least one cell of a multicellular orunicellular organism comprising administering directly to themulticellular or unicellular organism: a) a transposon comprising theexogenous nucleic acid, wherein the exogenous nucleic acid is flanked byone or more of any of the aforementioned inverted repeat sequences thatare recognized by any of the aforementioned proteins; and b) any one ofthe aforementioned proteins to excise the exogenous nucleic acid from aplasmid, episome, or transgene and integrate the exogenous nucleic acidinto the genome. In some embodiments, the protein of b) is administeredas a nucleic acid encoding the protein. In some embodiments, thetransposon and nucleic acid encoding the protein of b) are present onseparate vectors. In some embodiments, the transposon and nucleic acidencoding the protein of b) are present on the same vector. When presenton the same vector, the portion of the vector encoding the hyperactivetransposase is located outside the portion carrying the inserted nucleicacid. In other words, the transposase encoding region is locatedexternal to the region flanked by the inverted repeats. Put another way,the transposase encoding region is positioned to the left of the leftterminal inverted repeat or to the right of the right terminal invertedrepeat. In the aforementioned methods, the hyperactive transposaseprotein recognizes the inverted repeats that flank an inserted nucleicacid, such as a nucleic acid that is to be inserted into a target cellgenome.

In some embodiments, the multicellular or unicellular organism is aplant or animal. In some embodiments, the multicellular or unicellularorganism is a vertebrate. In some embodiments, the vertebrate animal isa mammal, such as for example, a rodent (mouse or rat), livestock (pig,horse, cow, etc.), pets (dog or cat), and primates, such as, forexample, a human.

The methods described herein can be used in a variety of applications inwhich it is desired to introduce and stably integrate an exogenousnucleic acid into the genome of a target cell. In vivo methods ofintegrating exogenous nucleic acid into a target cell are known. Theroute of administration of the transposon system to the multicellular orunicellular organism depends on several parameters, including: thenature of the vectors that carry the system components, the nature ofthe delivery vehicle, the nature of the multicellular or unicellularorganism, and the like, where a common feature of the mode ofadministration is that it provides for in vivo delivery of thetransposon system components to the target cell(s). In certainembodiments, linear or circularized DNA, such as a plasmid, is employedas the vector for delivery of the transposon system to the target cell.In such embodiments, the plasmid may be administered in an aqueousdelivery vehicle, such as a saline solution. Alternately, an agent thatmodulates the distribution of the vector in the multicellular orunicellular organism can be employed. For example, where the vectorscomprising the subject system components are plasmid vectors,lipid-based such as a liposome, vehicles can be employed, where thelipid-based vehicle may be targeted to a specific cell type for cell ortissue specific delivery of the vector. Alternately, polylysine-basedpeptides can be employed as carriers, which may or may not be modifiedwith targeting moieties, and the like (Brooks et al., J. Neurosci.Methods, 1998, 80, 137-47; and Muramatsu et al., Int. J. Mol. Med.,1998, 1, 55-62). The system components can also be incorporated ontoviral vectors, such as adenovirus-derived vectors, sindbis-virus derivedvectors, retrovirus-derived vectors, hybrid vectors, and the like. Theabove vectors and delivery vehicles are merely representative. Anyvector/delivery vehicle combination can be employed, so long as itprovides for in vivo administration of the transposon system to themulticellular or unicellular organism and target cell.

The elements of the PiggyBac transposase system are administered to themulticellular or unicellular organism in an in vivo manner such thatthey are introduced into a target cell of the multicellular orunicellular organism under conditions sufficient for excision of theinverted repeat flanked nucleic acid from the vector carrying thetransposon and subsequent integration of the excised nucleic acid intothe genome of the target cell. As the transposon is introduced into thecell “under conditions sufficient for excision and integration tooccur,” the method can further include a step of ensuring that therequisite PiggyBac transposase activity is present in the target cellalong with the introduced transposon. Depending on the structure of thetransposon vector itself, such as whether or not the vector includes aregion encoding a product having PiggyBac transposase activity, themethod can further include introducing a second vector into the targetcell that encodes the requisite transposase activity, where this stepalso includes an in vivo administration step. Because of the multitudeof different types of vectors and delivery vehicles that can beemployed, administration can be by a number of different routes, whererepresentative routes of administration include, but are not limited to:oral, topical, intraarterial, intravenous, intraperitoneal,intramuscular, and the like. In some embodiments, the administering isadministering systemically. The particular mode of administrationdepends, at least in part, on the nature of the delivery vehicleemployed for the vectors that harbor the PiggyBac transposons system. Inmany embodiments, the vector or vectors harboring the PiggyBactransposase system are administered intravascularly, such asintraarterially or intravenously, employing an aqueous based deliveryvehicle, such as a saline solution.

The amount of vector nucleic acid comprising the transposon element, andin many embodiments the amount of vector nucleic acid encoding thetransposase, which is introduced into the cell is sufficient to providefor the desired excision and insertion of the transposon nucleic acidinto the target cell genome. As such, the amount of vector nucleic acidintroduced should provide for a sufficient amount of transposaseactivity and a sufficient copy number of the nucleic acid that isdesired to be inserted into the target cell. The amount of vectornucleic acid that is introduced into the target cell varies depending onthe efficiency of the particular introduction protocol that is employed,such as the particular in vivo administration protocol that is employed.

The particular dosage of each component of the system that isadministered to the multicellular or unicellular organism variesdepending on the nature of the transposon nucleic acid, e.g. the natureof the expression module and gene, the nature of the vector on which thecomponent elements are present, the nature of the delivery vehicle andthe like. Dosages can readily be determined empirically by those ofskill in the art. For example, in mice where the PiggyBac transposasesystem components are present on separate plasmids which areintravenously administered to a mammal in a saline solution vehicle, theamount of transposon plasmid that is administered in many embodimentstypically ranges from about 0.5 to 40 μg and is typically about 25 μg,while the amount of PiggyBac transposase encoding plasmid that isadministered typically ranges from about 0.5 to 25 μg and is usuallyabout 1 μg.

Once the vector DNA has entered the target cell in combination with therequisite transposase, the nucleic acid region of the vector that isflanked by inverted repeats, i.e. the vector nucleic acid positionedbetween the PiggyBac transposase-recognized inverted repeats, is excisedfrom the vector via the provided transposase and inserted into thegenome of the targeted cell. As such, introduction of the vector DNAinto the target cell is followed by subsequent transposase mediatedexcision and insertion of the exogenous nucleic acid carried by thevector into the genome of the targeted cell.

The subject methods may be used to integrate nucleic acids of varioussizes into the target cell genome. Generally, the size of DNA that isinserted into a target cell genome using the subject methods ranges fromabout 0.5 kb to 100.0 kb, usually from about 1.0 kb to about 60.0 kb, orfrom about 1.0 kb to about 10.0 kb.

The subject methods result in stable integration of the nucleic acidinto the target cell genome. By stable integration is meant that thenucleic acid remains present in the target cell genome for more than atransient period of time, and is passed on a part of the chromosomalgenetic material to the progeny of the target cell. The subject methodsof stable integration of nucleic acids into the genome of a target cellfind use in a variety of applications in which the stable integration ofa nucleic acid into a target cell genome is desired. Applications inwhich the subject vectors and methods find use include, for example,research applications, polypeptide synthesis applications andtherapeutic applications.

The present invention can be used in, for example, germline mutagenesisin a rat, mouse, or other vertebrate; somatic mutagenesis in a rat,mouse, or other vertebrate; transgenesis in a rat, mouse, or othervertebrate; and use in human gene therapy. In each of these, thehyperactive transposase can be delivered as DNA, RNA, or protein.

The hyperactive PiggyBac transposase system described herein can be usedfor germline mutagenesis in a vertebrate species. One method wouldentail the production of transgenic animals by, for example, pronuclearinjection of newly fertilized oocytes.

Typically, two types of transgenes can be produced; one transgeneprovides expression of the transposase (a “driver” transgene) in germcells (i.e., developing sperm or ova) and the other transgene (the“donor” transgene) comprises a transposon containing gene-disruptivesequences, such as a gene trap. The transposase may be directed to thegermline via a ubiquitously active promoter, such as the ROSA26(Gt(ROSA)26Sor), pPol2 (Polr2a), or CMV/beta-actin (CAG) promoters.Alternately, one may use a germline-restricted promoter, such as thespermatid-specific Protamine-1 (Prm1) promoter, for mutagenesisexclusively in developing sperm. In another embodiment, the germlinespecific promoter is a female-specific promoter (e.g., a ZP3 promoter).

To achieve mutagenesis in this scenario, one can breed driver and donortransgenic lines to create double-transgenic animals. In doubletransgenic animals, which contain both transgenes in their genome, thePiggyBac transposase expressed in germ cells catalyzes the excision ofthe transposon and mediates mobilization to another site in the genome.If this new site contains a gene, then gene expression or proteinproduction can be perturbed through a gene trap. The most effective genetraps consist of strong splicing signals, whereby disruption andcreation of a null allele is mediated through a strong splice acceptor.A strong splice acceptor can also create alleles of altered function(such as a dominant negative, dominant active, or gain of function).Alternately, expression is rendered ectopic, constitutive, or alteredthrough the use of a heterologous promoter and strong splice donor.

Mutagenesis occurs in the germline of double-transgenic animals (withboth driver and donor transgenes) and upon breeding double-transgenicanimals, mutant offspring with heritable and permanent mutations areproduced. Mutations can be generated by injection of a fertilized oocytewith transposase RNA or protein. Alternatively, the transposase (as DNA,RNA, or protein) is electroporated, transfected or injected intoembryonic stem cells, induced pluripotent stem cells, or spermatogonialstem cells. These mutations (transposon insertions) can be detected by,for example, Southern blot and PCR. The specific insertion sites withineach mutant animal can then be identified by, for example,linker-mediated PCR, inverse PCR, or other PCR cloning techniques. Someof the mutant animals identified via PiggyBac-mediated mutagenesis canserve as valuable models for studying human disease.

Somatic mutagenesis is very useful for discovering tumor suppressors andoncogenes in a model vertebrate animal, such as the rat. Suchexperiments are otherwise not possible in humans, but throughPiggyBac-driven mutagenesis, carcinogenesis can be triggered, much inthe same way that ionizing radiation triggers carcinogenesis through DNAdamage. With PiggyBac transposon-mediated insertional mutagenesis,however, mutations can easily be pinpointed through, for example, PCRcloning techniques. The mutations uncovered are often directly linked tothe cancer, and in a single animal, hundreds of such mutations can beidentified. This is incredibly valuable for linking specific genes ascausative agents (tumor suppressors and oncogenes) that are directlyinvolved in providing the growth and survival advantages inherent in adeveloping neoplasia.

For somatic mutagenesis, the transgenic strategy can be very similar tothat for germline mutagenesis, except the driver transgene providesexpression of the transposase in the tissue where carcinogenesis will betargeted. For example, the intestine-specific Villin (Vil 1) promotercan provide highly specific expression of the PiggyBac transposase fortargeted mutagenesis and carcinogenesis in the intestine and colon. Thisprovides a valuable gene-discovery system of colon cancer in whichoncogenes and tumor suppressors directly linked to colon cancer can beeasily and rapidly identified. The donor transgene would likely be abi-directional gene trap that can cause a loss of function, such as anull allele in either orientation, and a gain of function. Thegain-of-function parameter is achieved through the use of a constitutivepromoter, perhaps containing a strong enhancer sequence, whichover-expresses a trapped oncogene. The resulting tumors fromPiggyBac-mediated mutagenesis would likely contain both types ofmutations, and thus both tumor suppressors and oncogenes can beuncovered.

For gene therapy to be practical, one should achieve stable integrationof a therapeutic transgene in the genome of an afflicted tissue toprovide a long-term and cost-effective treatment. Viruses provideeffective gene delivery but are either highly immunogenic orcarcinogenic. The PiggyBac transposon can mediate gene delivery in atarget tissue with a much lower risk for immune reactions and cancer.The inherently low immunogenicity of the PiggyBac transposon is due toits simplicity; there are no coat proteins, no receptor molecules, andno extracellular components, but simply a single small enzyme thatinteracts with host factors to mediate transposon insertion. While thePiggyBac transposon shows a slight preference for inserting withingenes, this preference is much less pronounced that a retrovirus, whichhas a very high preference for inserting within transcriptional units.

To achieve a high-efficiency and low-immunogenic gene transfer intopatients, one can use synthetic compounds for delivering DNA into acell. Liposomes and other nanoparticles are sufficient for this task.For PiggyBac-mediated gene therapy, two plasmids can be delivered to thepatient: one that provides expression of the transposase (a driverplasmid), and another that provides the transposon containing atherapeutic transgene (the donor plasmid). These DNAs can be complexedwith liposomes and administered via parenteral injection. Upon enteringa cell the PiggyBac transposase may bind to the transposon in the donorplasmid, excise it, and then integrate it into the genome. Suchinsertions will be stable and permanent. The driver and donor plasmidswill eventually be lost by cellular- and host-defense mechanisms, butany genome-integrated PiggyBac transposons, containing the therapeutictransgene, will be stable and permanent modifications. The transientnature of these plasmids also curtails excessive transposition, and thusminimizes the risk of carcinogenesis.

The present invention also provides methods of generating a transgenic,non-human vertebrate comprising in the genome of one or more of itscells a PiggyBac transposon which comprises nucleotide sequence that,when integrated into the genome, modifies a trait in the transgenic,non-human vertebrate, comprising: introducing ex vivo into a non-humanvertebrate embryo or fertilized oocyte a nucleic acid comprising aPiggyBac transposon which comprises a nucleotide sequence that, whenintegrated into the genome, modifies a trait in the transgenic,non-human vertebrate, and, within the same or on a separate nucleicacid, a nucleotide sequence encoding a PiggyBac transposase; implantingthe resultant non-human vertebrate embryo or fertilized oocyte into afoster mother of the same species under conditions favoring developmentof the embryo into a transgenic, non-human vertebrate; and, after aperiod of time sufficient to allow development of the embryo into atransgenic, non-human vertebrate, recovering the transgenic, non-humanvertebrate from the mother; thereby generating a transgenic, non-humanvertebrate comprising in the genome of one or more of its cells PiggyBactransposon.

The present invention also provides methods of mobilizing a PiggyBactransposon in a non-human vertebrate, comprising: mating a firsttransgenic, non-human vertebrate comprising in the genome of one or moreof its germ cells a PiggyBac transposon, wherein the PiggyBac transposoncomprises a nucleotide sequence, that when integrated into the genome,modifies a trait in the transgenic, non-human vertebrate, with a secondtransgenic, non-human vertebrate comprising in the genome of one or moreof its germ cells a nucleotide sequence encoding a PiggyBac transposaseto yield one or more progeny; identifying at least one of the one ormore progeny comprising in the genome of one or more of its cells boththe PiggyBac transposon and the nucleotide sequence encoding thePiggyBac transposase, such that the PiggyBac transposase is expressedand the transposon is mobilized; thereby mobilizing the PiggyBactransposon in a non-human vertebrate. The first and second transgenic,non-human vertebrates can be generated according to any of the methodsdescribed herein or known to those skilled in the art.

In some methods of transgenesis, transgenes are introduced into thepronuclei of fertilized oocytes. For some animals, such as micefertilization is performed in vivo and fertilized ova are surgicallyremoved. In other animals, particularly bovines, it is suitable toremove ova from live or slaughterhouse animals and fertilize the ova invitro. In vitro fertilization permits a transgene to be introduced intosubstantially synchronous cells at an optimal phase of the cell cyclefor integration (not later than S-phase). Transgenes are usuallyintroduced by microinjection (see, U.S. Pat. No. 4,873,292). Fertilizedoocytes are cultured in vitro until a pre-implantation embryo isobtained containing about 16-150 cells. Methods for culturing fertilizedoocytes to the pre-implantation stage are described by, for example,Gordon et al., Methods Enzymol., 1984, 101, 414; Hogan et al.,Manipulation of the Mouse Embryo: A Laboratory Manual, C.S.H.L. N.Y.(1986) (mouse embryo); Hammer et al., Nature, 1985, 315, 680; Gandolfiet al, J. Reprod. Pert., 1987, 81, 23-28; Rexroad et al., J. Anim. Sci.,1988, 66, 947-953; Eyestone et al., J. Reprod. Pert., 1989, 85, 715-720;Camous et al., J. Reprod. Fert., 1984, 72, 779-785; and Heyman et al.,Theriogenology, 1987, 27, 5968. Pre-implantation embryos can be storedfrozen for a period pending implantation. Pre-implantation embryos aretransferred to an appropriate female resulting in the birth of atransgenic or chimeric animal depending upon the stage of developmentwhen the transgene is integrated. Chimeric mammals can be bred to formtrue germline transgenic animals. The PiggyBac transgenes describedabove are introduced into nonhuman mammals. Most nonhuman mammals,including rodents such as mice and rats, rabbits, sheep, goats, pigs,and cattle.

Alternately, transgenes can be introduced into embryonic stem cells (ES)or SS cells, or iPS cells, etc. These cells are obtained frompreimplantation embryos cultured in vitro (Bradley et al., Nature, 1984,309, 255-258). Transgenes can be introduced into such cells byelectroporation or microinjection. Transformed ES cells are combinedwith blastocysts from a nonhuman animal. The ES cells colonize theembryo and in some embryos form the germ line of the resulting chimericanimal (Jaenisch, Science, 1988, 240, 1468-1474). Alternately, ES cellscan be used as a source of nuclei for transplantation into an enucleatedfertilized oocyte giving rise to a transgenic mammal.

For production of transgenic animals containing two or more transgenes,such as in embodiments where the PiggyBac transposon and PiggyBactransposase components of the invention are introduced into an animalvia separate nucleic acids, the transgenes can be introducedsimultaneously using the same procedure as for a single transgene.Alternately, the transgenes can be initially introduced into separateanimals and then combined into the same genome by breeding the animals.Alternately, a first transgenic animal is produced containing one of thetransgenes. A second transgene is then introduced into fertilized ova orembryonic stem cells from that animal.

Transgenic mammals can be generated conventionally by introducing bymicroinjecting the above-described transgenes into mammals' fertilizedeggs (those at the pronucleus phase), implanting the eggs in theoviducts of female mammals (recipient mammals) after a few additionalincubation or directly in their uteri synchronized to thepseudopregnancy, and obtaining the offspring.

To find whether the generated offspring are transgenic, many procedures,such as dot-blotting, PCR, immunohistological, complement-inhibitionanalyses, and the like, can be used. The transgenic mammals generatedcan be propagated by conventionally mating and obtaining the offspring,or transferring nuclei (nucleus transfer) of the transgenic mammal'ssomatic cells, which have been initialized or not, into fertilized eggsof which nuclei have previously been enucleated, implanting the eggs inthe oviducts or uteri of the recipient mammals, and obtaining the cloneoffspring.

Transformed cells and/or transgenic organisms, such as those containingthe DNA inserted into the host cell's DNA, can be selected fromuntransformed cells and/or transformed organisms if a selectable markeris included as part of the introduced DNA sequences. Selectable markersinclude, for example, genes that provide antibiotic resistance; genesthat modify the physiology of the host, such as for example greenfluorescent protein, to produce an altered visible phenotype. Cellsand/or organisms containing these genes are capable of surviving in thepresence of antibiotic, insecticides or herbicide concentrations thatkill untransformed cells/organisms or producing an altered visiblephenotype. Using standard techniques known to those familiar with thefield, techniques such as, for example, Southern blotting and polymerasechain reaction, DNA can be isolated from transgenic cells and/ororganisms to confirm that the introduced DNA has been inserted.

In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner.

Throughout these examples, molecular cloning reactions, and otherstandard recombinant DNA techniques, were carried out according tomethods described in Maniatis et al., Molecular Cloning—A LaboratoryManual, 2nd ed., Cold Spring Harbor Press (1989), using commerciallyavailable reagents, except where otherwise noted.

EXAMPLES Example 1 Using a MoMuLV System of Transduction for ScreeningVariants

In many ways, an efficient retroviral method of transduction is idealfor screening individual mutants within a single cell. Retroviralvectors derived from the MoMuLV retrovirus contain elements forexpression and packaging of the RNA genome, but lack genes that enablereplication. These vectors contain viral long terminal repeats (LTRs), apsi packaging signal that regulates encapsidation of the RNA, a site forcloning the cDNA of interest, and typically, a selectable marker such asthe neomycin phosphotransferase gene (Neo). These viral vectors aretransfected (as plasmid DNA) into a special packaging cell line thatexpresses genes necessary for encapsidation of the RNA genome intoinfectious virions. The virus is then harvested by collecting thesupernatant from transfected packaging cells. Upon infecting asusceptible target cell one or more viral particles fuse with the cellmembrane and are uncoated. The nucleocapsid (uncoated virion) enters thenucleus, where it is reverse-transcribed into DNA, and integrates intothe genome as a permanent proviral insertion. Since the LTRs of viralvectors act as a strong promoter in many cells, the cDNA of choice isexpressed. The proviral insertion cannot produce more infectious virusparticles.

If one produces a cDNA library within a retroviral vector, one canassess the function of distinct cDNAs in individual cells containingproviral insertions. This can be accomplished through transienttransfection of a packaging cell line to produce a retroviral library.During a transient transfection, individual cells may have thousands ofdifferent retroviral vectors, but each virion produced contains twoidentical copies of the same RNA (Flynn et al., J. Virol., 2004, 78,12129-39; and Flynn et al., Virology, 2006, 344, 391-400). If onegenerates a retroviral library containing 10⁷ cfu per ml, then millionsof target cells can be infected with one ml of viral supernatant suchthat each cell, on average, is infected with one virion. This wouldrepresent a multiplicity of infection (MOI) of one. This allows one toscreen millions of cDNA variants as single proviral insertions withineach cell. The true power of a diverse high-titer retroviral library isreflected in the efficiency of gene delivery to individual target cells.

Example 2 Screening Large Libraries of Variants

Large libraries of DNA fragments, with greater than 10¹⁰ variants, areeasily produced through error-prone PCR methods orpolymerase/DNase-based recombination methods. Because of the developmentof new ultra-efficient strains of electrocompetent E. coli, about 10⁶ to10⁷ variants can be amplified from ligated plasmid DNA. A difficultylies in creating a cell-based biological system that can thenindividually screen each mutant. The methods described herein enable oneto screen of millions of transposase variants. This process isfacilitated by the improved efficiency of MoMuLV vectors and viruspackaging lines. A packaging line derived from 293T cells, termedPlatinum-E (Plat-E), which produces high-titer virus (>10⁷ cfu/ml) fromtransiently transfected plasmid DNA is used herein (Morita et al., GeneTher., 2000, 7, 1063-6). Transient transfection is desired to maintainlibrary diversity. The level of efficiency of the Plat-E cell lines isachieved through enhanced expression of the gag-pol and env retroviralgenes in a cell-line (293T) that is easily transfected at highefficiency. This cell line out-performs two previously designedpackaging cell lines, Bosc23 and Phoenix-Eco. The Plat-E cell lineproduces replication-defective ecotropic viruses, which only infectmouse and rat cells via the ecotropic retroviral receptor, and are,thus, quite safe.

While reagents and techniques allow the generation of large diverseretroviral libraries, each distinct variant should be easilydiscriminated in an appropriate cell-based assay. For selection of ahyperactive transposase, one would want to select a transposase that canrapidly and efficiently mobilize a transposon from one genomic locationto another. Thus, for a given experimental period a hyperactivetransposase should yield a greater number of transposon integrations percell vs. a mediocre transposase. Described herein is a specialtransposon-based selection system, which when integrated, yieldseither: 1) a green fluorescent protein (GFP) signal proportional to thenumber of integrations per cell, or 2) variable resistance to the toxicalkaloid colchicine, which is likewise proportional to the number ofintegrations per cell.

Example 3 Measuring Transposase Activity Through Copy-Number DependentExpression

To assay the activity of transposase variants within each library, theintensity of GFP fluorescence as a read-out of transposase efficiencywas used. This can be accomplished by using polyA-trap genetraptransposons, which have been constructed. Two versions of the transposonhave been created, each containing the PiggyBac ITRs recognized by thePiggyBac transposase, termed BII-sd2GFP and BII-sMdr1 (see, FIG. 1).These genetraps express either a destabilized GFP (d2GFP) or humanmultidrug resistance gene (Mdr I or ABCB I) upon integration within agene and upstream of a polyadenylation (polyA) signal, and has beendesigned to yield copy-number dependent expression. Thus, a hyperactivetransposase drives the insertion of multiple copies of the transposon,yielding more GFP signal or a higher Mdr1 gene dosage. The GFP proteinis destabilized by a C-terminal PEST domain, which causes rapidturnover. This yields a larger dynamic range for measuring copy-numberdependent expression of GFP; this allows one to easily discriminatebetween low-expressers (low copy-number) and high-expressers (highcopy-number). High copy number transposition events of the BII-sMdr1transposon is achieved through stringent drug selection with themicrotubule-depolymerizing toxic alkaloid called colchicine. Higherdoses of colchicine require concomitantly greater expression levelsofMdr1 for cell survival (Kane et al., Gene, 1989, 84, 439-46; Kane etal., Mol. Cell. Biol., 1988, 8, 3316-21; and Kane et al., Biochem.Pharmacal., 2001, 62, 693-704). Mdr1 is a glycoprotein transporter thatconfers resistance to a variety of drugs, including chemotherapeuticcompounds, by reducing intracellular levels of the drug (Metz et al.,Virology, 1995, 208, 634-43; Pastan et al., Proc. Natl. Acad. Sci. USA,1988, 85, 4486-90; and Ueda et al., Proc. Natl. Acad. Sci. USA, 1987,84, 3004-8).

Copy number-dependent expression of the polyA trap is conferred byseveral additional components. Within the transposon, the reporter (GFPor Mdr1) is driven by a constitutive promoter that lacks CpGdinucleotides, and thus cannot be silenced by methylation. In addition,the genetrap is flanked by the core insulator from the chickenbeta-globin locus control region hypersensitive site IV (cHSIV)(Burgess-Beusse et al., Proc. Natl. Acad. Sci. USA, 2002, 99, 16433-7;Chung et al., Proc. Natl. Acad. Sci. USA, 1997, 94, 575-80; and Chung etal., Cell, 1993, 74, 505-14). The cHSIV insulator insulates against anyadjacent enhancers to reduce variability in expression(enhancer-blocking activity). This cHSIV element also prevents theencroachment of gene-silencing heterochromatin (insulator activity) thatcould silence expression (Burgess-Beusse et al., Proc. Natl. Acad. Sci.USA, 2002, 99, 16433-7; Chung et al., Proc. Natl. Acad. Sci. USA, 1997,94, 575-80; and Chung et al., Cell, 1993, 74, 505-14). These propertiesconfer copy number-dependent expression of an integrated transgene.However, because this genetrap lacks a polyA signal, the reporter onlygenerates a stable transcript following integration into a gene. ThepolyA trap components include an internal ribosomal entry site (IRES)from the encephalomyocarditis virus downstream of the GFP/Mdr1 openreading frame (ORF). The IRES prevents degradation of hybrid mRNAspecies expressed following a polyA trap event, which occurs through apoorly understood process called nonsense mediated mRNA decay (Shigeokaet al., Nucleic Acids Res., 2005, 33, e20). Immediately following theIRES is a splice donor (SD) from the exon1/intron1 boundary of theadenovirus type 2 (Ad2) late major transcript, which enables thereporter transcript to splice with the splice acceptor of a trappedexon. To prevent expression of integrations that do not occur within amulti-exon gene, an mRNA instability signal from the 3′ untranslatedregion (UTR) of the mouse Csj3 gene was also included, which causesactive deadenylation and degradation of the mRNA transcript. The use ofan mRNA instability signal from the Csf3 gene was effectively employedfor reducing transcript levels in a previous design of a polyA trap(Ishida et al., Nucleic Acids Res., 1999, 27, e35).

All of these components prevent any significant expression prior tomobilization (or upon mobilization into intergenic regions distal tosplice acceptors and polyA signals) but permits expression whenintegration occurs within genes, in a copy number-dependent manner.Prior to mobilization the transposon can be introduced into cells in twomanners: 1) as a multi-copy tandem array (concatemer) integrated intothe genome of cells at an intergenic region (where the polyA trap doesnot have any genes nearby for splicing and transcript stabilization) or2) as a transfected circular plasmid.

For the BII-sd2GFP transposon one can easily distinguish the absence of(or weak) GFP expression produced by an unmobilized transposon from theincreased GFP expression that occurs when the transposon integrates intoa gene. Fluorescence is therefore a surrogate marker of transposaseactivity and enables cell sorting by FACS. The efficient mobilization ofthe BII-sMdr1 transposon is easily screened by treating cells withincreasing amounts of colchicine; only PiggyBac transposases that canmobilize multiple copies of the BII-sMdr1 transposon are isolatedfollowing stringent selection with high doses of colchicine. Those cellsexhibiting a high level of GFP fluorescence or tolerating high doses ofcolchicine can then be collected.

Referring to FIG. 1, these transposons contain components for drivingexpression of either the destabilized GFP (d2GFP) or human Mdr1 proteinupon insertion within a gene, and upstream of a polyadenylation signal.Expression of either cDNA is driven by a CpG-less promoter that consistsof a mouse cytomegalovirus (CMV) enhancer and a basal promoter from thehuman Ef1α gene. Downstream of the d2GFP or Mdr1 cDNA is an IRES thatprevents non-sense mediated decay of hybrid transcripts. The splicedonor (SD) is from the human Adenovirus type 2 late major transcript,and efficiently permits splicing with 3′ trapped exons. An mRNAinstability signal from the Csf3 gene (zigzag) minimizes transcriptlevels when no polyA trap occurs. The chicken hypersensitive site IV(cHSIV) from the beta globin locus provides enhancer-blocking activityand insulation from adjacent heterochromatin, which promotes consistentcopy-number dependent expression. The inverted terminal repeats (ITRs,arrows) from the PiggyBac transposons are recognized and providemobilization by the respective transposases.

In some embodiments, five individual PB mutations were assessed fortranspositional activity in a flow-cytometry based assay in which EGFPfluorescence serves as a read-out of transposition into the genome asdescribed below. One PB mutation, M226F, yielded a greater than 2-foldincrease in the number of EGFP-positive cells.

To select for high levels of transposase activity, a high-throughputfluorescent activated cell-sorting (FACS) assay that measures theintensity of EGFP fluorescence as a read-out of transposase efficiencywas developed. This was accomplished by using a polyA-trap genetraptransposon, called the sEGFP transposon, which was constructed atTransposagen (see, FIG. 2A). sEGFP transposons were created that areflanked by the PB, inverted terminal repeats (ITRs). This genetrapexpresses EGFP upon integration within a gene and upstream of apolyadenylation (polyA) signal, and has been designed to yieldcopy-number dependent expression. Thus, in principle, a hyperactivetransposase will drive the insertion of multiple copies of thetransposon, yielding more EGFP expression, and thus a brighterfluorescence signal. Copy number-dependent and polyA-dependentexpression is conferred by several additional components (see, FIG. 2A).

Referring to FIG. 2A, the sEGFP Poly-A trap transposon is shown.Expression of the EGFP cDNA is driven by a CpG-less promoter (consistingof a mouse cytomegalovirus (CMV) enhancer and a basal promoter from thehuman Ef1α gene). Downstream of the EGFP ORF is an IRES that preventsnon-sense mediated decay of hybrid transcripts following a successfulgenetrap event. The splice donor (SD) is from the human Adenovirus type2 late major transcript, and permits efficient splicing when integrationoccurs 5′ of an exon. An mRNA instability signal from the Csf3 gene(zigzag) destabilizes untrapped transcripts. The cHSIV insulator (blueovals) promotes consistent copy-number dependent expression. Theinverted terminal repeats (ITRs, arrows) from the SB, TniPB, or TcBtransposons provide mobilization by the respective transposases.Referring to FIG. 2B, FACS analysis is shown. 2.5×10⁶ NIH3T3 cells wereelectroporated with 2.5 μg of the sEGFP transposon flanked by piggyBacITRs, along with 1.5 μg of an expression plasmid containing a PBtransposase (pCMV-PBoM226F) or an empty vector (pCMV empty), and thenassayed by flow cytometry 72 hours later. Most EGFP+ cells were between10- to 100-fold below the detection maximum.

Example 4 Identifying Rational Substitutions within the PiggyBacTransposase

To determine the phylogenetic deviation of the PiggyBac transposases,PiggyBac-like polypeptide sequences from the following species (with theindicated GenBank accession numbers) were analyzed for phylogeneticcomparison: Trichoplusia ni (AAA87375.2), Xenopus tropicalis(BAF82022.1), Bombyx mori (BAD11135.1), Heliothis virescens(ABD76335.1), Macdunnoughia crassigna (ABZ85926.1), Strongylocentrotuspurpuratus (XP_797885.2), Culex pipiens quinqefasciatus(XP_001869225.1), Daphnia pulicaria (AAM76342.1), Helicoverpa armigera(ABS18391.1), Dania rerio (XP_699416.2), Nasonia vitripennis(XP_001599370.1), Bactrocera dorsalis (AF289123), Tak(fugu rubripes(scaffold_270, 208447-210099), Dania rerio (XP_699416.1), Gasterosteusaculeatus (Chr:groupXX, 10624991-10626727), Ciona savignyi (reftig_140,9946-11658), Ciona intestinalis (NW_001955008.1, 51209-52888, andNW_001955804.1,1016-2667), Anopholes gambiae (NZ_AAAB02008849,2962684-2964410), Tribolium castaneum (NW_001092821.1, 1981685-1983397),and Myotis lucifugus (GeneScaffold_410, 157651-159369). These elementsrepresent invertebrate and vertebrate PiggyBac-like transposases thateach contains a critical DDD motif (D268, D346, D447) and a tryptophanresidue (W465), which are all necessary for Trichoplusia ni PiggyBacenzyme activity. Protein sequences were aligned by the ClustalW method.Individual amino acid positions of the PiggyBac transposase sequencewere deemed divergent if no more than five PiggyBac-like sequences (fromspecies other than Trichoplusia ni) shared the PiggyBac amino acid (orhighly-similar amino acid) with Trichoplusia ni (T. ni) at a givenposition, when no fewer than seven non-T. ni transposases contained theidentical (or highly-similar) amino acid for that position. Suchcommonly shared amino acids at a given position among thesePiggyBac-like transposases represent a “consensus” amino acid sequence.In addition, at least twice as many species must contain the consensusamino acid, compared to the number of species (including T. ni) thatshare an identical non-consensus amino acid. The rational substitutionof individual amino acids was deduced from divergent positions, suchthat PiggyBac sequences were reverted, or restored, to the consensus.These rational substitutions are illustrated along a schematic of thePiggyBac transposase protein in FIG. 3A and FIG. 4A.

Example 5 Generating Diverse Permutations of the Rational Substitutions

To assess the interaction and possible cooperation between theserational substitutions, a method called the staggered extension process(StEP), a PCR-based DNA recombination strategy entailing frequenttemplate switching, was used. StEP creates random permutations of twoclosely related sequences through template switching, made possible byallowing only brief periods of primer extension (FIG. 3B). A diversecollection of variants is obtained after many cycles of priming,extension, and template switching. The StEP method is employed toshuffle two templates: (1) a codon-optimized PiggyBac transposase (PBo)sequence containing some or all of these 32 rational substitutions(termed PB-var) along with (2) a codon-optimized (PBo) cDNA sequencethat codes for the wild type T. ni PiggyBac amino acid sequence (FIG.3C). For StEP, equimolar concentrations of the PBo and the PB-var DNAare combined in 8 to 12 separate reactions containing a primer pair thatflanks two unique SfiI sites and the PiggyBac transposase sequence. Tominimize mutations introduced by PCR and keep diversity limited to thedescribed substitutions, Phusion (NEB) high fidelity polymerase wasused. Thermocycling uses this program: 98° C. for 60 seconds, 98° C. for15 seconds, and 56° C. for 5 seconds, for 198 cycles. The short 56° C.step incorporates primer annealing and a very brief period of polymeraseextension, producing small fragments, which after denaturation thenrandomly anneals to homologous regions (template switching). 198 cyclesare desired to generate full-length hybrid fragments at a length of 2Kb. To eliminate the original plasmid DNA, which is dam-methylated atGATC sites, PCR reactions are digested with DpnI, run on a 1% agarosegel, extracted, pooled, and if DNA yield is too low, a standardhigh-fidelity PCR is performed on this template. DNA is digested withSfiI and ligated into the retroviral expression vector pLXIN (Clontech)that has been modified by adding two unique and compatible SfiI sites.This ligation is then purified by agarosc gel electrophoresis and 50 to100 ng of DNA is electroporated each into 4 aliquots of DH1OB Mega-X(Invitrogen) electrocompetent cells, then recovered by shaking at 37° C.for 45 minutes. Serial dilutions (10-2, 10-3, 10-4) of eachtransformation are plated onto LB-diversity. Ampicillin to determine thenumber of colony forming units (cfu) per ml and to estimate potentialApproximately 5×10⁶ cfu from the library is then amplified in semi-solidLB agar and DNA isolated using the Pure Yield Plasmid Midiprep Kit(Promega). Prior to DNA isolation, a small aliquot of the amplifiedtransformation is plated on LB agar plus Ampicillin, and 24 coloniespicked and sequenced to assess diversity and recombination frequencybetween each substitution.

A schematic describing the PB mutations is shown in FIG. 4B; this PB-varsequence was shuffled with a wildtype PBo sequence. After shuffling, DNAlibraries were ligated via directional SfiI sites into a modified pLXIN(Clontech) vector. Following transformation of each library into Mega-Xelectrocompetent DH1OB cells (Invitrogen), 5×10⁶ cfu were amplified forthe PB library, in semi-solid agar. Plasmid DNA was isolated and used totransfect Platinum-E cells for retrovirus production.

Upon cloning into a retroviral vector, transduction of the library intoNIH-3T3 cells enables each individual transposase to be assayed in asingle cell, if one adjusts the multiplicity of infection (MOT) suchthat each cell receives, on average, one functional proviral insertion.A hyperactive transposase will drive the mobilization of multipletransposon insertions per cell. The sEGFP transposon, which is capableof driving copy-number dependent expression, serves as a surrogateread-out of transposase efficiency, as determined by the intensity ofEGFP fluorescence. Transduced cells expressing a library of variants canthus be functionally sorted using FACS. An alternate strategy byselecting for puromycin-resistance using a transposon that contains apuromycin acetyltrasferase (PAC) expression cassette, called BII-SVPuro(for PB), was also devised. The PAC expression cassette also containsSV40 promoter/enhancer and polyadenylation (pA) signals. After thelibrary screens, the proviral insertion can then be PCR amplified fromgenomic DNA from these sorted cells, and a secondary library generated.By repeating this process, one can enrich for the most hyperactivevariants.

Referring to FIG. 4A, the PiggyBac transposase with alterations spreadthroughout the polypeptide sequence; mutations include single amino acidsubstitutions and two double amino acid substitutions (VF240KL andYY327RV). The nuclear localization signal (NLS, black box) is located atthe C-terminus of the polypeptide. The catalytic domain (shaded gray)spans a large central core, and contains the catalytic DDD box, which ishighlighted in red. Referring to FIG. 4B, StEP PCR entailed successiverounds of brief polymerization, followed by denaturation, and templateswitching through promiscuous annealing. Referring to FIG. 4C, shufflingbetween two PiggyBac transposase sequences was accomplished by combiningthe wild-type PBo sequence with a mutated sequence (containing varioussubstitutions) followed by a PCR amplification with very brief periodsof polymerase activity (StEP PCR). Template switching produces multipleiterations, and thus, a collection of diverse variants.

Referring again to FIG. 4, two substitutions were planned at position591 (Q591P or Q591R), but ultimately the Q591R substitution wasdiscarded. Essential aspartates (red) residues make up the catalytic DDDbox. The nuclear localization signal (NLS, black box) is located nearthe C-terminus. Equal amounts of two templates (1 part M185L, 1 partA187G, and 1 part M185LIA187G versions of PB-var) and 3 parts WT PBowere combined and StEP PCR produced shuffled sequences. PCR productswere digested with SfiI to create a retroviral library to be ultimatelyscreened by drug resistance selection or FACS.

Example 6 Creating and Screening the Retroviral Library

After diversity has been evaluated and, the library has been amplified,Plat-E packaging cells (Cell Biolabs) in 10 cm dishes are transientlytransfected with 7.5 μg of the amplified and purified plasmid libraryusing LipoD293 Transfection Reagent (SignaGen). Viral supernatant isthen collected 48 hours later, gently filtered through a 0.45 μm filter,and mixed with polybrene (8 μg/ml final concentration). Viral titers areassessed by infecting NIH-3T3 cells with serial dilutions (10-1, 10-2,10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶) of the viral supernatant in 6-wall plates. Atiter between 5×10⁵ and 1×10⁷cfu/ml is typical.

To assay the activity of variants within each library, GFP intensity asa surrogate marker of transposase efficiency is used. First, theBTT-sd2GFP transposon is integrated into the genome of NIH-3T3 cells asa concatemer repeat, to establish a source of the transposon in a nativechromatin environment. This is a standard procedure accomplished bytransfecting NIH-3T3 cells with the BII-sd2GFP transposon along withone-tenth (molar ratio) the amount of a plasmid containing a puromycinor hygromycin selectable marker. Stable lines are selected by pickingindividual drug-resistant colonies that are not GFP-positive, asassessed by examination under an inverted fluorescent microscope. Thecopy number of the sd2GFP transposon can then be evaluated byquantitative PCR (QPCR). Clones with at least copies are retained forfurther study. Each cell line is then evaluated for transposition bytransfection of an expression vector containing the PB transposase.Successful mobilization of the BII-sd2GFP transposon is then evaluatedby flow cytometry on a Becton Dickinson FACScaliber (BD Biosciences).

Retroviral libraries are then used to infect approximately 5×10⁶NIH-3T3-sd2GFP cells at an MOI of one. After 8 hours, the medium ischanged, and after 16 hours the medium is changed again, and thenincubated for an additional 48 hours. Cells are sorted for the brightestGFP fluorescence (if at least 100-fold over background) on a FACSVantageSE (BD Biosciences). Genomic DNA is isolated from these cells and thetransposase coding sequence within proviral insertions is amplified byPCR using Phusion polymerase (NEB). PCR products are digested with SfiIand ligated into the pLXIN retroviral vector and a second retrovirallibrary produced again as above. This first generation (G1) retrovirallibrary is used to infect NIH-3T3-sd2GFP cells and the process repeated(in triplicate) to produce subsequent generations (G2, G3, and up to G4,with three preparations/sorts per generation) until a library yieldshomogenous hyperactivity as assessed by FACS. A refining process occursthrough this repeated cycling, which culminates in a small collection ofhyperactive transposases. No more than four generations are analyzed.

Proviral insertions from the final population of sorted cells areamplified by PCR and clones sequenced to identify each hyperactivetransposase. The hyperactivity of isolated PB transposases is thencompared to wildtype T. ni PBo by FACS assays in HEK293T cells and in achromosomal transposition assay in HeLa cells (Baus et al., MolecularTherapy, 2005, 12, 1148-1156; Ivies et al., Cell, 1997, 91, 501-10;Zayed et al., Mol. Ther., 2004, 9, 292-304; and Yant et al., Mol. Cell.Biol., 2004, 24, 9239-47). In the chromosomal transposition assay, a Neocassette in the transposon confers G418-resistance while the transposaseis expressed from a separate plasmid.

Example 7 Initial Screening of the Primary (GO) Shuffled TransposaseLibraries

To screen PB libraries using the transposons containing the PACexpression cassettes, approximately 1.2×107 NIH-3T3 cells wereelectroporated with the respective SVPuro transposon and split betweenfour T175 flasks. Twenty-four hours after electroporation, cells wereinfected with the PB retroviral libraries. Puromycin selection yieldedhundreds of thousands of surviving cells from each library. In theabsence of transposase expression (i.e., following infection with avirus lacking a transposase open reading frame), no cells survivedpuromycin selection. After four days of selection with puromycin,genomic DNA was isolated and the provirus was PCR amplified with PhusionHot Start (Finnzymes) using primers that flank the unique pair of SfiIsites adjacent to the coding region of the transposase. The amplifiedtransposases were subcloned into the pLXIN vector for production of thesecondary (G1) transposase libraries. The generation of a G1 library forthe PB transposase was accomplished. Table 1 shows a quantitativesummary of the current stage of screening for the primary (GO) andsecondary (G1) libraries.

TABLE 1 # of G0 # of G1 Clones Retroviral Clones Retroviral # ofPossible Amplified Library Amplified Library Shuffled # of (GO Titer (G1Titer Mutations Iterations library) (cfu/ml) library) (cfu/ml) 21 2.1 ×10⁶ 5 × 10⁶ 3 × 10⁶ 2.1 × 10⁶ 5 × 10⁵ 13 8,192 1 × 10⁶ 8 × 10⁶ No dataNo data

Example 8 Production of Hyperactive PB Transposases (M185L, A187G)

Using phylogenetic conservation to suggest rational substitution, twomutant PiggyBac transposases, M185L and A187G, which each demonstratehyperactivity in a FACS-based transposition assay, have been generatedand tested. The activity of these mutant transposases with thepolyA-trap transposon, BII-sEGFP, which is similar to the BII-sd2GFPtransposon except an enhanced (non-destabilized) GFP (EGFP) codingsequence has been substituted for the d2GFP sequence, is illustratedherein. The EGFP protein enables more sensitive detection of singletransposition events in single cells. HEK293T cells were transfectedwith plasmids containing the PB transposon (B11-sEGFP) and each mutantPB transposase. Approximately 3×10⁵ HEK293T cells were transfected in6-well plates with calcium-phosphate and the GFP fluorescence wasanalyzed 72 hours later by FACS (FIG. 5). With the wild-typecodon-optimized PB (PBo) transposase, about 4% of cells are GFP-positive, whereas only about 1% are positive without the transposase(FIG. 5A). If only GFP+++ “bright” cells, which exhibit at least100-fold greater fluorescence over background, are examined then thisdifference is more pronounced, with about 5-fold more GFP “bright” cellsproduced versus the wild-type PBo transposase. The mutant PiggyBactransposases, M185L and A187G, yield increased mobilization of theBII-sEGFP transposon, as measured by the number of GFP positive cells(FIG. 5). The M185L mutant produces about 50% more GFP+ cells (FIG. 5A),and about 30% more GFP+++ cells (FIG. 5B), compared to wild-type PBo.The A187G mutant produces approximately 70% more GFP+ cells (FIG. 5A),and about 100% more GFP+++ cells (FIG. 5B), compared to wild-type PBo.These assays illustrate an increased ability of these mutanttransposases to mobilize the BII-sEGFP transposon. This hyperactivitycould be due to an enhanced stability of the transposase, an increase inthe catalytic efficiency, and/or an augmented preference for integrationwithin genes (which would yield more GFP signal). Any of these featureswould be desirable for performing mutagenesis in vertebrate or mammaliancells.

Referring to FIG. 5A, flow cytometry assay measuring GFP fluorescenceproduced when the polyA-trap transposon (BII-sEGFP) inserts into thegenome, upstream of a polyadenylation signal. Without the transposase(No Phase), very few fluorescent cells were observed, whereas thewild-type PBo produces a four-fold increase in GFP+ cells. The M185L andA187G mutant transposases exhibited enhanced activity, yielding a sixand seven-fold increase, respectively, in the number of GFP-positivecells. Referring to FIG. 5B, a similar trend was observed when examiningGFP+++ “bright” cells, which exhibited at least 100-fold greater GFPfluorescence over background. Remarkably, the A187G mutant PBtransposase yielded approximately 10-times as many GFP+++ cells than the“No Pbase” control, and 2-times as many GFP+++ cells as the wild-typePBo transposase.

Example 9 Identification of Hyperactive PB Mutations

Several individual mutations were characterized for their ability toconfer hyperactivity to the PB transposases: Q591P, M185L, A187G, andM226F. Quantification of transposition by the flow cytometry assay inHEK293T cells revealed that all of these mutations conferred some degreeof hyperactivity, with the M226F yielding over 2-fold greater number ofGFP-positive cells (FIG. 6A). The M226F mutation also yielded nearlytwice the mean fluorescence of all gated cells, as compared to wildtypePBo (not shown), suggesting that the number of insertions mobilized, percell, was greater for PboM226F.

The primary (G0) library for PB was initially screened for the abilityto mobilize a PB transposon containing a PAC expression cassette(BII-SV-Puro), which confers puromycin resistance in NIH-3T3 cells. 24clones from the ensuing G1 library were sequenced to glean some idea ofmutation abundance. In the unselected library (G0), the sequence of 77clones was observed; the abundance of most mutations was close to 50%,as expected, except for M185L and A1X7G, which were expected to bepresent in 331% of all variants due to our strategy for manuallyshuffling these particular mutations (FIG. 6B).

Comparison of the G0 and G1 clones revealed that the M185L, A187G,F200W, M226F, M282Q, and G165S mutations were the six most-enrichedmutations among all 21 shuffled substitutions. The F200W mutation, whilenot yet functionally assessed for hyperactivity, was one of the two moststrongly suggested rational substitutions, by phylogeny (a tryptophanwas observed at position 200 for 15 out of 20 aligned PB transposases).While the sampling size is incredibly small for the analysis of a largelibrary, the enrichment of three out of four known hyperactive mutationsappears very significant (FIG. 6B). In addition, restriction digestanalysis with BsrGI, which is diagnostic for the M282Q mutation,revealed that the M282Q mutation is clearly present in the largemajority of the G1 plasmid library (data not shown). In all, it appearsthat puromycin selection was successful in not only selecting forfunctionally active variants, but was also likely effective forselecting hyperactive variants.

Referring to FIG. 6A, Several hyperactive PB mutations were identifiedvia quantification of EGFP positive cells by flow cytometry, which isindicative of genomic transposition of the BTT-sEGFP polyA-traptransposon. The M185L, A187G, and M226F mutations (boxed in red) appearsignificantly more active than wildtype PBo. Referring to FIG. 6B, thesix most highly enriched mutations for each library after puromycinselection, among 77 and 24 sequenced clones, respectively, is depicted.The same three known hyperactive mutations (boxed in red) are alsoenriched in the puro-selected library. The F200W substitution wasstrongly suggested by phylogenetic alignment of 20 PB transposases(boxed in black). While the sampling size was small, restriction digestdiagnosis of M282Q abundance (asterisk) in the plasmid library confirmedclear enrichment of M282Q.

TABLE 2 PiggyBac Transposase Mutations PiggyBac Mutation Nucleic AcidSEQ ID NO: Protein SEQ ID NO: S3N 93 94 BOV 95 96 A46S 3 4 A46T 5 6 182W97 98 S103P 99 100 R119P 7 8 C125A 9 10 C125L 11 12 G165S 101 102 Y177K13 14 Y177H 15 16 F180L 17 18 F180I 19 20 F180V 21 22 M185L 23 24 Al87G25 26 F200W 27 28 V207P 29 30 V209F 31 32 M226F 33 34 L235R 35 36 V240K37 38 F241L 39 40 P243K 41 42 N258S 103 104 M282Q 43 44 L296W 45 46L296Y 47 48 L296F 49 50 M298L 51 52 M298A 53 54 M298V 55 56 P311I 57 58P311V 59 60 R315K 61 62 T319G 63 64 Y327R 65 66 Y328V 67 68 C340G 69 70C340L 71 72 D421H 73 74 V436I 75 76 M456Y 77 78 L470F 79 80 S486K 81 82M503L 83 84 M503I 85 86 V552K 87 88 A570T 89 90 Q591P 105 106 Q591R 107108

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, and the like) cited in the present application isincorporated herein by reference in its entirety.

What is claimed is:
 1. A hyperactive transposase protein comprising atleast 90% sequence identity to SEQ ID NO: 2, and comprising an aminoacid substitution at positions 30, 165 and 282, wherein the substitutionat position 30 is a substitution of a Valine for an Isoleucine (I30V),wherein the substitution at position 165 is a substitution of a Serinefor a Glycine (G165S), wherein the substitution at position 282 is asubstitution of an amino acid (X) for a Methionine (M282X), the aminoacid (X) selected from the group consisting of a Leucine (L), anIsoleucine (I), a Valine (V), and an Alanine (A), and wherein thehyperactive transposase protein is capable of mediating transposonintegration at a higher frequency when compared to a wild typetransposase having the sequence of SEQ ID NO:
 2. 2. A nucleic acidencoding the hyperactive transposase protein of claim
 1. 3. A vectorcomprising the nucleic acid of claim
 2. 4. A cell comprising the vectorof claim
 3. 5. A cell comprising the nucleic acid of claim
 2. 6. A cellcomprising the hyperactive transposase protein of claim
 1. 7. A methodof integrating an exogenous nucleic acid into the genome of a cellcomprising: introducing the exogenous nucleic acid and the hyperactivetransposase protein of claim 1 or a nucleic acid encoding thehyperactive transposase protein into a cell under conditions sufficientfor integration of the exogenous nucleic acid to occur.
 8. The method ofclaim 7, wherein a vector comprises the nucleic acid encoding thehyperactive transposase protein and wherein the vector comprises atleast one regulatory element operatively linked to the nucleic acidsequence encoding the hyperactive transposase protein.
 9. The method ofclaim 8, wherein the vector is a plasmid, a liposome, a virus or ananoparticle.
 10. The method of claim 8, wherein the vector is ananoparticle.
 11. The method of claim 9, wherein the virus is anadenovirus-derived vector, sindbis-virus derived vector,retrovirus-derived vector or a hybrid vector.
 12. The method of claim 9,wherein the virus is an adenovirus-derived vector.
 13. The method ofclaim 12, wherein the adenovirus-derived vector comprises long terminalrepeats, a psi packaging signal, a cloning site, and a sequence encodinga selectable marker.
 14. The method of claim 7, wherein a carriercomprises the nucleic acid encoding the hyperactive transposase proteinand wherein the carrier comprises at least one regulatory elementoperatively linked to the nucleic acid sequence encoding the hyperactivetransposase protein.
 15. The method of claim 14, wherein the carriercomprises a polylysine-based peptide.
 16. The method of claim 7, whereina transposon comprises an insertion site for the exogenous nucleic acid,wherein the insertion site is flanked by a first inverted repeatsequence comprising a sequence at least about 90% sequence identity toSEQ ID NO: 91 and/or a second inverted repeat sequence comprising asequence at least about 90% sequence identity to SEQ ID NO:
 92. 17. Thehyperactive transposase protein of claim 1, wherein the substitution atposition 282 is a substitution of a Valine (V) for a Methionine (M282V).